Stem cells and their environment, the so-called niche, are critical components that sustain not only proper tissue homeostasis, but also diseased states such as cancer. The inability to follow the same cells over time in an intact animal is a bi challenge that has greatly limited our understanding of what goes awry during normal tissue homeostasis and the cellular behaviors exhibited in the initial stages of tumor growth. Specifically, this roadblock has hindered our ability to understand both the role of specific cells and how their location contributes to their growth, whether it be normal or cancerous. For the first time, we are now capable of addressing these questions since my lab has pioneered the use of live imaging in skin stem cell regeneration and established an approach that allows us to follow the same cells over time in an intact, live mouse. By these approaches we have learned that first, the location where stem cells reside influences their ability to contribute to growth. Second, as a consequence of a perturbation, such as loss of a stem cell pool, other cells can acquire a new ability to contribute to growth that they did not possess previously. Third, the induction of oncogenic mutations in clones, such as mutations that stabilize the Wnt effector ?-catenin, will drive aberrant growths ultimately leading to tumor formation through the recruitment of non cell-autonomous wild-type cells. However, it is still unclear 1) what signaling mechanisms regulate the aberrant cellular behaviors that will eventually cause tumor development, 2) whether a cell's position within a tissue influences different tumoral outcomes, and 3) what early cell behaviors are adopted by stem and other cell types that lead to tumor development. To identify the aberrant stem cell/progenitor activities that cause cancer, we are using two contrasting human skin tumors; a benign and a malignant form, which we can faithfully recapitulate in a mouse model. We plan to 1) utilize cutting-edge exome and RNA sequencing approaches to identify target gene mutations associated with tumoral epithelial and stromal cell populations and 2) to assess the role of these genes towards cancer by in vivo overexpression and shRNA knockdown in the mouse via in utero injection and subsequent functional validation using our in vivo imaging approach. Together, these approaches will allow us to understand how novel and established target genes alter stem cell behaviors and identify the initial sequential steps that lead to malignant tumor growth. Understanding the mechanisms and signaling pathways underlying the role of stem cells in cancer holds great promise to transform current therapeutic strategies. Our plan aims to identify the relevant cells and genes that are responsible for skin tumor initiation, which may prove relevant for an extended application to other types of cancer.